Ubiquitination Via the E3 Ligase TRAF3 by Targeting ASC for K63

MAVS Promotes Inflammasome Activation by Targeting ASC for K63-Linked Ubiquitination via the E3 Ligase TRAF3

This information is current as Kai Guan, Congwen Wei, Zirui Zheng, Ting Song, Feixiang of September 24, 2021. Wu, Yanhong Zhang, Ye Cao, Shengli Ma, Wei Chen, Quanbin Xu, Weiwei Xia, Jun Gu, Xiang He and Hui Zhong J Immunol 2015; 194:4880-4890; Prepublished online 6 April 2015;

doi: 10.4049/jimmunol.1402851 Downloaded from http://www.jimmunol.org/content/194/10/4880

Supplementary http://www.jimmunol.org/content/suppl/2015/04/04/jimmunol.140285 Material 1.DCSupplemental http://www.jimmunol.org/ References This article cites 35 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/194/10/4880.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

MAVS Promotes Inﬂammasome Activation by Targeting ASC for K63-Linked Ubiquitination via the E3 Ligase TRAF3

Kai Guan,*,1 Congwen Wei,*,1 Zirui Zheng,*,1 Ting Song,*,1 Feixiang Wu,† Yanhong Zhang,* Ye Cao,* Shengli Ma,* Wei Chen,† Quanbin Xu,* Weiwei Xia,‡ Jun Gu,‡ Xiang He,* and Hui Zhong*

Stringent control of inflammasome signaling pathway is important for maintaining immunological balance, yet the molecular mechanisms responsible for its tight regulation are still poorly understood. In this study, we found that the signaling pathway dependent on mitochondrial antiviral signaling protein (MAVS) was required for the optimal activation of apoptosis-associated specklike protein (ASC)–dependent inflammasome. In particular, TNFR-associated factor 3 was found to be a direct E3 ligase for ASC. Ubiquitination of ASC at Lys174 was critical for speck formation and inflammasome activation. Deficiency in MAVS or TNFR- associated factor 3 impaired ASC ubiquitination and cytosolic aggregates formation, resulting in reduced inflammasome response Downloaded from upon RNA virus infection. This study has identified a previously unrecognized role of MAVS in the regulation of inflammasome signaling and provided molecular insight into the mechanisms by which ubiquitination of ASC controls inflammasome activity through the formation of ASC specks. The Journal of Immunology, 2015, 194: 4880–4890.

nflammatory reactions in response to pathogen infection are associated specklike protein (ASC), and procaspase-1 (9). The http://www.jimmunol.org/ mediated by locally released cytokines and chemokines, such inflammasome-initiating event is the recognition of intracellular I as macrophage-released IL-1b and IL-18 (1). Deregulated damage-associated molecular patterns derived from pathogens inflammatory response has been linked to autoimmune diseases (pathogen-associated molecular patterns) or hosts (danger or stress (2), including inflammatory bowel diseases (3, 4), vitiligo (5), signals) by cytosolic NLRs. NLRs undergo ATP-dependent olig- gouty arthritis (6), and diabetes (7, 8). Determining the mecha- omerization in response to damage-associated molecular pattern nisms that regulate IL-1b maturation and secretion is therefore of recognition and recruit ASC by PYD–PYD interaction. Sub- considerable interest. The activation of caspase-1 is essential for sequently, caspase-1 is recruited through the C-terminal caspase the processing of pro–IL-1b and pro–IL-18, and the secretion of recruitment domain (CARD) of ASC, which is essential for its their mature biologically active forms (3). A critical step in the activation (3). Macrophages deficient in ASC are impaired in by guest on September 24, 2021 activation of caspase-1 is the assembly of a large protein complex activating caspase-1 upon infection and tissue damage, empha- containing the NOD-like receptors (NLRs), adapter apoptosis- sizing a central role of ASC in the activation of inflammatory caspases (1, 10, 11). *State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnol- Cytosolic viral nucleic acids can be recognized by a family of ogy, Beijing 100850, People’s Republic of China; †Department of Hepatobiliary DexD/H box RNA helicases including retinoic acid–inducible Surgery, Affiliated Tumor Hospital of Guangxi Medical University, Nanning gene I (RIG-I) and melanoma differentiation-associated gene 5 530021, People’s Republic of China; and ‡Department of Biochemistry and Molec- ular Biology, College of Life Sciences, Peking University, Beijing 100871, People’s (MDA5). Both RIG-I and MDA5 contain CARD(s) that interact Republic of China with the CARD-containing protein mitochondrial antiviral sig- 1K.G., C.W., Z.Z., and T.S. contributed equally to this work. naling protein (MAVS) upon binding to uncapped dsRNA (12). Received for publication November 14, 2014. Accepted for publication March 8, Binding of RIG-I and MDA5 to MAVS through CARD–CARD 2015. homotypic interactions leads to the stabilization and accumulation This work was supported by the Basic Research Program of China (Grants of MAVS homodimer (13), which can then directly recruit ubiq- 2012CB518900 and 2010CB529403), the National Natural Science Foundation of China (Grants 31471325, 31470850, 31300637, 31170029, 31270911, 31270800, uitin ligase TNFR-associated factor (TRAF) 3/5/6 and activate 81071771, 81401641, 30725035, 30930103, and 31100960), and the National Sci- their E3 ligase activity (14–16). Active TRAF3/5/6, in turn, may ence and Technology Major Program (Grant 2011ZX09102-010-02). mediate the recruitment and activation of TBK1 through scaffold Address correspondence and reprint requests to Prof. Jun Gu, Xiang He, and Hui molecules, such as NEMO and TANK (17). Zhong, Beijing Institute of Biotechnology, 27 Tai-Ping Road, Haidian, Beijing 100850, China. E-mail addresses: [email protected] (J.G.), [email protected] The RIG-I pathway adaptor MAVS was initially believed to exert (X.H.), and [email protected] (H.Z.) effects on immune signaling mainly by bridging the interaction The online version of this article contains supplemental material. between RIG-I and downstream effectors, such as TBK1, IKKε, Abbreviations used in this article: ASC, apoptosis-associated specklike protein; and STING. However, emerging evidence indicates that MAVS BMDM, bone marrow–derived macrophage; CARD, C-terminal caspase recruitment also plays important roles in controlling inflammatory responses. domain; CPPD, calcium pyrophosphate dihydrate; DSS, disuccinimidyl suberate; IRF, a b b IFN regulatory factor; MAVS, mitochondrial antiviral signaling protein; MDA5, mela- For instance, the induction of IFN- , TNF- , IL-6, and IL-1 noma differentiation-associated gene 5; MEF, mouse embryonic fibroblast; MOI, mul- by bacterial and viral RNAs is dependent on MAVS (18–20). tiplicity of infection; MSU, monosodium urate; NLR, NOD-like receptor; PLA, Moreover, MAVS-deficient mice are highly susceptible to dis- proximity ligation assay; poly(dA:dT), polydeoxyadenylic-polydeoxythymidylic acid; poly(I:C), polyinosinic-polycytidylic acid; RIG-I, retinoic acid–inducible gene I; uccinimidyl suberate–induced colitis (18). These results indicate siRNA, small interfering RNA; TRAF, TNFR-associated factor; VSV, vesicular a cell-intrinsic role of MAVS in IL-1b production. The production stomatitis virus; WT, wild-type. of IL-1b requires NF-kB–dependent synthesis of pro–IL-1b and Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 inflammasome-dependent caspase-1 activation. Previous reports www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402851 The Journal of Immunology 4881 show that MAVS is essential for RIG-I–induced production of IL- QuikChange Mutagenesis kit (Invitrogen). Transient transfections were 1b via modulating NF-kB activation but dispensable for caspase-1 performed with Lipofectamine 2000 (Invitrogen) and INTERFERin (Poly- activation (21), although this remains controversial. Several recent plus) according to the manufacturer’s instructions. reports suggest that MAVS is required for optimal inflammasome Immunoprecipitation and immunoblot analysis activation by noncrystalline NLRP3 activators and polyinosinic- Cell lysates were prepared in lysis buffer (50 mM Tris-HCl [pH 7.5], 1 mM polycytidylic acid [poly(I:C)] (22), and is dispensable for NLRP3 PMSF, 1 mM DTT, 10 mM sodium fluoride, 10 mg/ml aprotinin, 10 mg/ml activation induced by ATP, nigericin, and particulate matter (23). leupeptin, and 10 mg/ml pepstatin A) containing 1% Nonidet P-40. Soluble Instead of focusing on MAVS-dependent NLRP3 mitochondrial lo- proteins were subjected to immunoprecipitation with different Abs. In calization, in this article, we provide evidence that MAVS is in- addition, an aliquot of the total lysates (5%, v/v) was included as control. volved in optimal inflammasome function upon RNA viral infection Samples were clarified, denatured with SDS buffer, and boiled for 10 min; separatedbySDS-PAGE;andtransferredtopolyvinylidenefluoride by binding ASC. MAVS is capable of stabilizing ASC and inducing membranes. The membranes were immunoblotted with primary Abs and its cytosolic speck formation, associated with the activity of the proteins detected with appropriate secondary Abs conjugated to HRP inflammasome via recruiting an E3 ligase TRAF3 to ASC. Ubiq- followed by ECL (Perkin Elmer Life Sciences). 174 uitination of ASC at Lys via TRAF3 is critical for speck forma- RNA interference tion and inflammasome activation. Deficiency in MAVS or TRAF3 impaired ASC ubiquitination and cytosolic speck formation, and The following small interfering RNA (siRNA) oligonucleotides were ob- 9 9 hence reduced inflammasome response upon viral infection. The tained from Invitrogen: MAVS, 5 -CCACCUUGAUGCCUGUGAA-3 ; TRAF3, 59-AGAGUCAGGUUCCGAUGAU-39. The negative control siRNA MAVS–TRAF3–ASC axis thus defines a new signaling cascade for was obtained from Invitrogen. ASC speck formation and “fine-tuning” of inflammasome activation. Downloaded from In situ proximity ligation assay Materials and Methods Fixed and permeabilized cells were incubated overnight at 4˚C with the following pairs of primary Abs: rabbit anti-ASC (Alexis) together with Mice mouse mAb to MAVS (Cell Signaling) or mouse mAb to NLRP3 (Alexis). MAVS2/2 mice on a 129/Sv/C57BL/6 background were maintained in The cells were washed and allowed to react to a pair of proximity probes specific pathogen-free conditions and were used at 6–9 wk of age. All the (Olink Bioscience). The rest of the in situ proximity ligation assay (PLA) animal experimental procedures were approved by the Animal Ethics and protocol was performed according to the manufacturer’s instructions. The http://www.jimmunol.org/ Research Committee of Beijing Institute of Biotechnology. cells were examined by fluorescence microscopy (Olympus), and the Duolink Image Tool (Olink Bioscience) was used for quantitative analysis. Cell culture Immunofluorescence microscopy Bone marrow–derived macrophages (BMDMs) were prepared as previously described (24). Bone marrow was flushed from femurs and tibia of Cells grown in LabTek (Fisher Scientific) chambers were fixed for 10 min in mice and was plated in RPMI-1640 supplemented with 30% L929 su- 4% paraformaldehyde, followed by permeabilization with 0.1% Triton pernatant containing macrophage-stimulating factor, glutamine, and so- X-100 in PBS for 15 min. The cells were then incubated for 1 h in blocking dium pyruvate. THP-1 cells were maintained in RPMI-1640 medium buffer (1% BSA in PBS) followed by incubation overnight with primary containing 10% (v/v) FBS (Gibco) and antibiotics. THP-1 cells were Abs. Next, cells were washed three times for 10 min each in PBS, then differentiated into macrophages by treatment for 3 h with 50 nM PMA incubated for 1 h with secondary Abs. Alexa Fluor 594–conjugated anti- by guest on September 24, 2021 (Sigma-Aldrich). TRAF32/2 mouse embryonic fibroblasts (MEFs), MAVS2/2 mouse or rabbit IgG and Alexa Fluor 488–conjugated anti-mouse or rabbit MEFs, and HEK293T cells were grown in DMEM (Invitrogen) supplemented IgG were used (Invitrogen). DNA was visualized by DAPI (Sigma-Aldrich) with 10% heat-inactivated FBS (Hyclone), 2 mM L-glutamine, 100 U/ml staining. Images were acquired using a Zeiss LSM510 confocal micro- 3 penicillin, and 100 mg/ml streptomycin at 37˚C under 5% CO2. scope (Zeiss) through a 60 oil fluorescence objective. Reagents and Abs Caspase-1 activity assay Reagents were from the following suppliers: LPS, monosodium urate (MSU), Cells were directly lysed in caspase lysis buffer (10 mM HEPES [pH 7.4], calcium pyrophosphate dihydrate (CPPD), and Flagellin (Invitrogen); 25 mM NaCl, 0.25% Triton X-100, and 1 mM EDTA) at 4˚C for 15 min. 3pRNA, ATP, poly(I:C), and polydeoxyadenylic-polydeoxythymidylic acid The lysates were centrifuged at 16,000 3 g for 10 min, and the supernatant [poly(dA:dT)] (Sigma-Aldrich). Commercial Abs were from the following was normalized for protein content. Samples were incubated with 2 mM suppliers: anti-ASC, anti-NLRP3, anti–caspase-1–p10, anti-Ubiquitin, and fluorogenic caspase-1 substrate Ac-YVAD-pNA at 37˚C for 1 h. The anti-Myc Abs (Santa Cruz); anti-MAVS and anti–IL-1b Abs (Cell Signaling protease activity was continuously measured by monitoring the release of Technology); and anti-Flag, anti–a-Tubulin, and anti-HA Abs (Sigma- free pNA at 37˚C (405 nm) and expressed as arbitrary fluorescence units Aldrich). The ELISA kits for human and mice IL-1b were purchased from per minute. Dakewe Biotech. Chemical cross-linking Inflammasome activation For detection of ASC oligomerization, pellets from whole-cell lysates were Cells were plated at a density of 1 3 105 per well in 24-well plates. Culture cross-linked with disuccinimidyl suberate (Sigma-Aldrich) for 15 min at medium was replaced with Opti-MEM (Invitrogen) before stimulation or 37˚C. The reaction was stopped by the addition of quenching solution at infection. PMA-differentiated THP-1 cells and BMDMs were primed for a final concentration of 20 mM Tris-HCl (pH 7.5) for 10 min at room 4 h with 1 mg/ml LPS and stimulated with CPPD (200 mg/ml), MSU temperature. Samples were then fractionated by SDS-PAGE and immu- (10 mg/ml), or ATP (2.5 mM). For delivery into the cytosol of macro- noblotted with anti-ASC Ab. phages, Flagellin, poly(dA:dT), and 3pRNA were introduced into primed In vivo ubiquitination assay macrophages through the use of Lipofectamine LTX (Invitrogen). Cells were infected with vesicular stomatitis virus (VSV) at a multiplicity of Cells were harvested 24 h after transfection, and whole-cell lysates were infection (MOI) of 10. After the indicated time of incubation, extracellular prepared in a 1% Nonidet P-40 lysis buffer supplemented with 0.1% virus was removed by washing cells twice with serum-containing media. protease inhibitor mixture (Roche) and 10 mM deubiquitinase inhibitor N-ethylmaleimide (Sigma-Aldrich). Soluble proteins were immunopreci- Plasmids pitated with 1 mg anti-Flag or anti-ASC Ab. Polyubiquitination was Vectors and Epitope Tagging of Flag-tagged MAVS, Myc-tagged MAVS, detected using anti-HA or anti-ubiquitin Ab. Flag-tagged TRAF3, Myc-tagged TRAF3, Flag-tagged ASC, and GFP- Influenza virus infection of BMDMs tagged ASC were expressed by cloning the genes into respective pcDNA3-based vectors. Flag-MAVS mutation, Flag-ASC, and GFP-ASC Influenza virus A/PR/8/34 (H1N1) was propagated in the allantoic cavity of mutation plasmids were made based on the pcDNA3-Flag-MAVS, 10-d-old embryonated specific pathogen-free chicken eggs and was mouse pcDNA3-Flag-ASC, and pEGFP-N2-ASC plasmids, respectively, using adapted by a minimum of six serial passages through mice. The viral titers 4882 MAVS CONTROLS ASC SPECK FORMATION were determined by standard plaque assay on confluent monolayers of MAVS reduced the amount of dimerized and oligomerized ASC in MDCK cells. Harvested BMDMs were washed twice with RPMI 1640 THP-1 cells (Fig. 1F). These results indicate that MAVS is re- 3 6 medium, seeded into 6-well plates (1 10 cells/well), and infected with quired for the formation of ASC specks. A/PR/8/34 at an MOI of 2. After 1-h viral adsorption at 37˚C, the inoculum was replaced with fresh media, and the cells were incubated at 37˚C. Cell- Because of the importance of ASC specks on activation of the free supernatant was harvested at select time points for viral titer and NLRP3 inflammasome, we next assessed the involvement of cytokine analysis. MAVS in IL-1b secretion. Infection with VSV induced robust b b In vivo influenza A virus infection and sampling IL-1 production in BMDMs from WT mice; by contrast, IL-1 production in response to the stimuli was severely impaired in Mice were anesthetized with Avertin (2, 2, 2-tribromoethanol) and infected 2/2 b 3 3 m MAVS BMDMs (Fig. 1G). Intracellular pro–IL-1 expression, intranasally with diluted virus (2 to 8 10 PFU) in 30 l endotoxin-free b b PBS. They were then either weighed and monitored for mortality daily for IL-1 cleavage, and IL-1 production in response to VSV infec- a period of 14 d or sacrificed at various intervals for sampling the cytokine tion were severely impaired in siMAVS THP-1 cells (Fig. 1H and levels, and serum was collected by cardiac puncture. 1I). Previous reports showed RIG-I–dependent type I IFN–in- For histopathologic examination, lungs were fixed by inflation and ducing inflammasome activation (27); we thus explored the immersion in 10% buffered formalin. To evaluate airway inflammation, we function of MAVS in direct inflammasome activation. SiMAVS subjected fixed lung slices to H&E staining. To assess viral titer, we removed, weighed, and homogenized whole THP-1 cells were infected with VSV for 8 h before treatment with lungs in PBS. Lung viral titers were determined in the supernatant as exogenous IFN-b. Notably, IFN-b treatment was not able to re- described earlier. store complete pro–IL-1b expression, IL-1b cleavage, and IL-1b Statistical analysis secretion in siMAVS cells (Fig. 1H and 1I). Congruence result showed that MAVS overexpression significantly increased caspase-1 Downloaded from 6 Data were presented as mean SEM. Two-tailed Student t test was used activation and caspase-1 catalysis activity (Fig. 1J and 1K), in- for evaluating statistical significance between groups. dicating a direct effect of MAVS on inflammasome activation. We also assessed the effects of MAVS on a series of stimuli that Results activated the NLRP3, NLRC4, and AIM2 inflammasome in mac- The formation of ASC specks in response to VSV infection rophages. Compared with control siRNA, treatment with MAVS- requires MAVS specific siRNA reduced IL-1b secretioninresponsetoNLRP3 http://www.jimmunol.org/ CARDs are protein interaction domains that play key roles in many activators 3pRNA (RIG-I agonist) and VSV (RIG-I and MDA5 cellular processes including inflammation and apoptosis (25). agonist), but not NLRP3 activators CPPD and MSU (crystals), MAVS contains an N-terminal CARD domain that interacts with NLRC4 activator Flagellin, or AIM2 activator poly(dA:dT) the CARDs of RIG-I, resulting in the activation of IFN-I signaling. (Supplemental Fig. 1F), indicating that MAVS plays an am- The adaptor protein of inflammasome complex ASC also contains plifying role in NLRP3-dependent IL-1b secretion specifically a CARD domain that connects specifically with procaspase-1 via induced by RNA virus infection. CARD–CARD interactions. Therefore, it is interesting to see the possible interactions of these two CARD-containing proteins. The MAVS modulates ASC protein stability specificity of ASC Ab was first confirmed by an ASC peptide To investigate how MAVS regulates inflammasome activation, we by guest on September 24, 2021 competition assay (Supplemental Fig. 1A). Immunofluorescence examined the effect of MAVS on ASC expression. The over- results showed that ASC colocalized with MAVS in mitochondria expression of MAVS led to a considerable increase of endogenous of THP-1 cells even in the absence of VSV infection (Fig. 1A). ASC (Fig. 2A). We next investigated the function of endogenous VSV infection led to the formation of small aggregates con- MAVS in ASC regulation. Expression of MAVS-specific short taining ASC and MAVS at 2 h postinfection, whereas MAVS siRNA oligos, but not control siRNA, decreased endogenous ASC failed to colocalize with ASC 12 h after VSV infection, when abundance in THP-1 cells (Fig. 2B). Interestingly, we did not nearly all of the ASC began to form into a single perinuclear detect any ASC expression in MAVS2/2 MEFs (Fig. 2C). Con- speck structure (Fig. 1A and Supplemental Fig. 1B). Further- sistent with this, immunofluorescence results showed that ASC more, we visualized ASC–MAVS complexes by an in situ PLA. staining was observed only in MAVS2/2 cells rescued success- We observed small spots of ASC–MAVS complex in resting fully with MAVS expression plasmid, whereas the staining of THP-1 cells, and these spots increased in abundance in response ASC intensity was nearly invisible in MAVS2/2 cells (Fig. 2D). to VSV infection (Fig. 1B and Supplemental Fig. 1C). Also, RT-PCR data showed that ASC mRNA abundance was not altered endogenous MAVS was found to be specifically coimmunopre- with MAVS expression (Fig. 2E), suggesting that MAVS upre- cipitated with endogenous ASC (Fig. 1C). Taken together, these gulates ASC by posttranscriptional modification. Unlike ASC, the results suggest the existence of ASC–MAVS complex during protein level of NLRP3 was unchanged by MAVS deficiency viral infection, and that the activated MAVS may participate in (Fig. 2C); however, NLRP3 displayed primarily nuclear distribu- the formation of ASC specks. tion in MAVS2/2 cells, whereas MAVS introduction led to ASC has been shown previously to form characteristic ASC NLRP3 redistribution in both nuclear and cytosolic compartments aggregates (“speckles”) that impact the activation of NLRP3 (Fig. 2F). To further confirm these observations, we analyzed inflammasome (26); thus, we would like to explore the possible nuclear and cytoplasmic fractions from MAVS2/2 cells by Western role of MAVS in the formation of ASC specks. MAVS-specific blotting for the subcellular distribution of ASC and NLRP3. As siRNA oligonucleotides, which reduced MAVS protein by ∼70%, showninFig.2G,NLRP3showedsteadylevelsinthenuclear were used (Supplemental Fig. 1D). Compared with sicontrol fractions of MAVS2/2 MEFs, whereas ASC was nearly undetect- THP-1 cells, the amounts of ASC aggregates induced by VSV able. However, compared with Flag-vector transfected control cells, infection were greatly reduced in siMAVS cells (Fig. 1D and MAVS-expressing cells showed higher levels of NLRP3 in the cy- Supplemental Fig. 1E). Compared with MAVS wild-type (WT) toplasmic fraction and lower levels of NLRP3 in the nuclear fraction, BMDMs, the ability of formatting ASC cytosolic aggregates in whereas the majority of the upregulated ASC was retained in the MAVS deficiency (MAVS2/2) BMDMs infected with VSV was nuclear fractions. These results indicate that MAVS may have a totally impaired (Fig. 1E). Indeed, immunoblot of the detergent- major role in regulating the stability of ASC and subcellular redis- resistant fraction post VSV infection showed that knockdown of tribution of NLRP3. The Journal of Immunology 4883 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 1. Formation of ASC specks in response to VSV infection requires MAVS. (A) Representative confocal immunofluorescence images of ASC colocalized with MAVS in THP-1 cells infected with VSV for the indicated time. Red represents MAVS; green represents ASC. (B) In situ PLA of ASC– MAVS complex in THP-1 cells infected with VSV for the indicated time. Red represents ASC–MAVS complexes; blue represents DAPI. (C) Immunoblot showing endogenous ASC–MAVS association in THP-1 cells. (D) Representative confocal immunofluorescence images of ASC colocalized with Mito- Tracker in THP-1 cells transfected with scrambled siRNA (siCtrl) or MAVS-specific siRNA oligos (siMAVS) and infected with VSV for 3 h. Red represents mitochondria; green represents ASC. (E) Representative confocal immunofluorescence images of ASC in WT or MAVS2/2 BMDMs cells infected with VSV for 3 h. Green represents ASC; Blue represents DAPI. (F) Immunoblot showing oligomerization level of ASC in THP-1 cells transfected with scrambled siRNA or MAVS-specific siRNA oligos and infected with VSV for the indicated time. (G) ELISA for IL-1b in supernatant of WT and MAVS2/2 BMDMs infected with VSV for 4 h. (H) ELISA for IL-1b in the cell-free supernatant. THP-1 cells were transfected with either control or MAVS-targeting siRNA and then infected with either mock or VSV for 8 h before treatment with either exogenous IFN-b (40 U/ml) or mock. (I) Immunoblot showing pro– IL-1b and cleaved IL-1b expression in cells from (H). (J) Immunoblot showing cleavage of procaspase-1 in THP-1 cells transfected with Myc-MAVS at indicated doses. (K) Measurement of caspase-1 activity in lysates of HEK293T cells transfected with indicated doses of MAVS. Imaging data are representative of images from three independent experiments. Cell-based studies were performed at least three times independently with comparable results. Data were presented as mean 6 SEM. Student t test was used for statistical analysis. Numbers below certain Western blots indicate relative levels determined by software-based quantification of the representative experiment shown. Scale bars, 10 mm(A, B, D, and E). **p , 0.01.

Identiﬁcation of TRAF3 as the E3 ligase for ASC ubiquitinated under physiological conditions of activation. Be- Endogenous ASC was immunoprecipitated with anti-ASC Ab at cause MAVS itself is not an E3 ubiquitin ligase, we reasoned that different times after VSV infection and ubiquitination was detected MAVS might function as an adaptor to recruit an E3 ubiquitin with anti-ubiquitin Ab, to investigate how MAVS regulates the ligase to ASC. To identify the E3 ubiquitin ligase(s) responsible stability of ASC. The appearance of a slower-migrating smear for ASC ubiquitination, we designed an assay to screen for IL-1b demonstrated that ASC ubiquitination occurred as early as 2 h production and ASC expression in HEK293T cells transfected postinfection and peaked at 4 h (Fig. 3A), indicating that ASC is with cDNA constructs from a sublibrary for human E3 ubiquitin 4884 MAVS CONTROLS ASC SPECK FORMATION Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 2. MAVS modulates ASC protein stability. (A) Immunoblot showing ASC level in THP-1 cells transfected with Myc-MAVS at indicated doses. (B) Immunoblot showing ASC level in THP-1 cells transfected with MAVS-specific siRNA oligos. (C) Immunoblot showing ASC and NLRP3 level in WT or MAVS2/2 MEFs. (D) Representative confocal immunofluorescence images of ASC in MAVS2/2 MEFs transfected with Flag-vector (Flag-V) or Flag- MAVS (WT). Red represents MAVS; green represents ASC; blue represents DAPI. (E) Quantitative RT-PCR for ASC expression in THP-1 cells transfected with Myc-MAVS at indicated doses. (F) Representative confocal immunofluorescence images of NLRP3 in MAVS2/2 MEFs transfected with Flag-vector (Flag-V) or Flag-MAVS (WT). Green represents NLRP3; red represents MAVS. (G) Subcellular fractionation of MAVS2/2 MEFs transfected with Flag- vector (Flag-V) or Flag-MAVS (WT). Nuclear (nuc) and cytosolic (cyto) fractions were analyzed for expression of NLRP3, ASC, and MAVS by immunoblot. Purity of the fractions was accessed by blotting for Lamin A (nuclear protein) and a-Tubulin (cytosolic protein). Imaging data are representative of images from three independent experiments. Cell-based studies were performed at least three times independently with comparable results. Data were presented as mean 6 SEM. Numbers below certain Western blots indicate relative levels determined by software-based quantification of the representative experiment shown. Scale bars, 10 mm(D and F). ligases. TRAF3 was found to promote both ASC stabilization and in THP-1 significantly impaired ASC cytosolic aggregates for- IL-1b production (Supplemental Fig. 2A–D). Coimmunoprecipi- mation induced by VSV (Fig. 3I and Supplemental Fig. 2E). tation experiments revealed that ASC interacted with endogenous These results suggest that TRAF3 is a novel E3 ubiquitin ligase TRAF3 in THP-1 cells (Fig. 3B). Furthermore, ASC was ubiq- for ASC that associates with inflammasome function. uitinated when HA-tagged WT ubiquitin or K63-only ubiquitin was expressed, whereas its ubiquitination was significantly re- MAVS engagement of TRAF3 and ASC is critical for duced upon expression of K48-only ubiquitin (Fig. 3C). TRAF3 inflammasome activation may enhance ASC levels by promoting K63 ubiquitination spe- To understand the mechanisms that link MAVS ligation to the cifically via E3 ligase activity, because TRAF3 C68A H70A or activation of TRAF3 and inflammasome activation, we investi- C53A C56A mutant (without E3 ligase activity) displayed com- gated the roles of the effectors in RIG-I-MAVS pathways. promised activity on ASC ubiquitination (Fig. 3D). Consistently, Consistent with the essential role of RIG-I in TRAF3 activation, cells transfected with TRAF3-specific siRNA showed decreased N-RIG-I and WT MAVS led to ASC upregulation. However, endogenous ASC expression (Fig. 3E), reduced pro–IL-1b ex- N-RIG-I K172R mutant or full-length RIG-I, which showed a pression, IL-1b cleavage (Fig. 3F), and IL-1b secretion post VSV complete loss of IFN-b promoter activation in unstimulated cells, infection (Fig. 3G). IL-1b secretion was also markedly reduced in failed to induce ASC upregulation. Moreover, the upregulation of TRAF3-deficient (TRAF32/2) MEFs in response to VSV infection ASC by TBK1 was aborted (Supplemental Fig. 2F), suggesting (Fig. 3H). Furthermore, transfection with TRAF3-specific siRNA that MAVS modulates ASC stability at the level of MAVS or The Journal of Immunology 4885

FIGURE 3. Identification of TRAF3 as the E3 ligase for ASC. (A) Immunoblot showing ASC ubiquitination level in THP-1 cells infected with VSV for the indicated time. (B) Immunoblot showing endogenous TRAF3–ASC association in THP-1 cells. (C and D) Immunoblot showing ubiquitination level of ASC in HEK293T cells transfected with the indicated plasmids and treated with MG132 (20 mM) for 12 h. (E) Immunoblot showing ASC level in THP-1 cells transfected with TRAF3-specific siRNA oligos. (F) Immunoblot showing intracellular pro– IL-1b and cleaved IL-1b expression in THP-1 cells transfected with scrambled siRNA or TRAF3-specific siRNA oligos. G b ( ) ELISA for IL-1 in the supernatant of Downloaded from THP-1 cells transfected with scrambled siRNA, MAVS-specific, or TRAF3-specific siRNA oligos infected with VSV for 3 h. (H) ELISA for IL-1b in the supernatant of WT and TRAF32/2 MEFs infected with VSV for 3 h. (I) Representative confocal immunofluorescence images of ASC in http://www.jimmunol.org/ THP-1 cells transfected with scrambled siRNA or TRAF3-specific siRNA oligos and infected with VSV for 3 h. Scale bar, 10 mm. Imaging data are representative of images from three independent experiments. Cell-based studies were performed at least three times independently with comparable results. Data were presented as mean 6 SEM. Student t test was used for statistical analysis. Numbers below certain by guest on September 24, 2021 Western blots indicate relative levels determined by software-based quantification of the representative experiment shown. K48-O, K48-only ubiquitin; K63-O, K63- only. **p , 0.01.

a level downstream of MAVS, but upstream of TBK1. Consis- bind TRAF3 (Fig. 4D). We then examined whether Q145N or tent with these findings, the elimination of MAVS expression by E457D mutant of MAVS affected the inflammasome signaling. siRNA abrogated MAVS-induced ASC upregulation, and elim- MAVS2/2 MEFs rescued by WT MAVS, Q145N, or E457D, ination of TRAF3 expression by siRNA inhibited ASC upreg- respectively, were infected with VSV followed by immunoflu- ulation mediated by overexpressedWTMAVS(Fig.4A).MAVS orescence to examine the ASC expression and ASC aggregates W517A dimerization mutant also failed to upregulate ASC, formation, or by ELISA to measure the IL-1b production. Cells indicating the importance of MAVS signaling pathway in reg- that were rescued by WT MAVS displayed increased ASC ulating inflammasome activation (Fig. 4A). We then assessed expression, increased number of ASC aggregates (Fig. 4E), the impact of MAVS and TRAF3 on ASC ubiquitination trig- and enhanced IL-1b secretion in response to VSV infection gered by VSV infection and found that ASC ubiquitination was (Fig. 4F). In contrast, cells that were rescued by Q145N or sharply reduced in siMAVS or siTRAF3 cells (Fig. 4B). Im- E457D failed to induce any ASC upregulation, aggregates for- portantly, coimmunoprecipitation assay of TRAF3 with ASC mation (Fig. 4E), or IL-1b secretion (Fig. 4F). Interestingly, demonstrated that TRAF3 immunoprecipitated more ASC in MAVS–NLRP3 colocalization induced by viral infection was sicontrol cells than in siMAVS cells after VSV infection (Fig. intact in cells transfected with Q145N or E457D mutant just as 4C), suggesting that TRAF3 recruitment to ASC is MAVS de- well as that with WT (Supplemental Fig. 2G). Taken together, pendent. MAVS engagement of TRAF3 induced by RNA virus infection is Reports showed that both N (aa 143–147) and C termini (aa critical for ASC speck formation and the subsequent ASC 450–468) of MAVS harbor potential TRAF3-binding site (28). inflammasome activation, whereas NLRP3 colocalization with By immunoprecipitation assay, we found that TRAF3 interacted MAVS induced by virus infection is independent of MAVS– with WT, but MAVS Q145N mutant or E457D mutant failed to TRAF3 signaling axis. 4886 MAVS CONTROLS ASC SPECK FORMATION

FIGURE 4. MAVS engagement of TRAF3 and ASC is critical for inflammasome activation. (A) Immunoblot showing ASC level in HEK293T cells transfected with Myc-ASC and Flag-vector (Flag-V), Flag- MAVS (WT), Flag-MAVS (W517A), MAVS- specific, or TRAF3-specific siRNA oligos. (B) Immunoblot showing ubiquitination level of ASC in THP-1 cells transfected with scrambled, MAVS-specific, or TRAF3- specific siRNA oligos infected with VSV for the indicated time. (C) Immunoprecipi- tation showing endogenous ASC–TRAF3 association in THP-1 cells transfected with scrambled siRNA or MAVS-specific siRNA

and infected with VSV for the indicated Downloaded from time. (D) Immunoprecipitation showing the interaction of MAVS or its mutants with TRAF3. (E) Representative confocal immunoﬂuorescence images of ASC in MAVS2/2 MEFs transfected with Flag- vector, Flag-MAVS, Flag-MAVS Q145N,

or Flag-MAVS E457D mutant, and infected http://www.jimmunol.org/ with VSV for 3 h. A total of 30 transfected MEF cells were analyzed. Red represents MAVS; green represents ASC; blue represents DAPI. Scale bar, 10 mm. (F) ELISA for IL-1b in the supernatant from (E). Im- aging data are representative of images from three independent experiments. Cell-based studies were performed at least three times independently with comparable results. Data were presented as mean 6 SEM. Student by guest on September 24, 2021 t test was used for statistical analysis. Numbers below certain Western blots indicate relative levels determined by software- based quantiﬁcation of the representative experiment shown. **p , 0.01.

Ubiquitination of ASC at Lys174 is indispensable for To definitively demonstrate the presence of two distinct com- inflammasome activation plexes upon viral infection, we fractionated cell extracts of To further reveal the possible TRAF3-mediated ubiquitination site(s) HEK293T cells on a size-exclusion column (HiPrep 16/60 Sephacryl in ASC, we tested 10 ASC KR mutants for their stability by intro- S-200 HR). Immunoblotting of fractions collected from chroma- ducing them with TRAF3 into HEK293T cells. Although TRAF3 had tography showed that both WTASC and ASC K174R mutant mainly ∼ little effect on the expression and ubiquitination level of ASC K174R coeluted with TRAF3 in fractions 23–29 (molecular mass 140 to mutant, it increased the expression and ubiquitination levels of other 60 kDa) in unstimulated cells. By contrast, a certain portion of WT . ASC KR mutants and WT ASC as well (Supplemental Fig. 3A and ASC was eluted in fractions 2–6 (molecular mass 670 kDa) upon 3B). Furthermore, ASC K174R induced significantly lower IL-1b VSV infection, which also contained TRAF3, whereas ASC K174R production than WT-ASC or other ASC KR mutants in the presence kept in the fractions 23–29, indicating that WT ASC may form ofTRAF3(SupplementalFig.3C).Itisofinteresttoexamine a larger complex with TRAF3 in the presence of VSV infection whether MAVS could also affect the protein stability of ASC K174R (Fig. 5B). Consistently, we observed an increased abundance of mutant. In contrast with WT ASC, the cellular abundance of ASC ASC–MAVS small spots in cells with ectopic WT ASC after VSV K174R mutant was unchanged under MAVS overexpression infection, but not in cells with K174R expression (Fig. 5C and (Supplemental Fig. 3D). The ability of ASC K174R mutant under- Supplemental Fig. 3E). Furthermore, VSV-activated cytosolic going ubiquitination induced by viral infection was also examined. aggregates were induced in WT ASC-transfected THP-1 cells, but A sharp reduction of ubiquitination induction was observed in ASC not in those transfected with ASC K174R mutant (Fig. 5D and K174R compared with WT ASC upon VSV infection, indicating that Supplemental Fig. 3F). Similar to the immunofluorescence result, Lys174 of ASC is its primary ubiquitination site (Fig. 5A). the ability of ASC K174R in VSV-induced IL-1b production was The Journal of Immunology 4887

FIGURE 5. Ubiquitination of ASC at Lys174 is indispensable for inﬂammasome activation. (A) Immunoblot showing ubiquitination level of ASC in HEK293T cells transfected with WT ASC or ASC K174R mutant and infected with VSV for 3 h. (B) Immunoblot showing TRAF3 and WT ASC or TRAF3 and ASC K174R level in fractionation of HEK293T cells infected with VSV for 3 h. Fractionation was monitored on a size-exclusion column (HiPrep 16/60 Sephacryl S 200HR). (C) In situ PLA of ASC–MAVS complexes in HEK293T cells Downloaded from transfected with WT ASC or ASC K174R mutant, infected with VSV for the indicated time. Red represents ASC–MAVS complex; blue represents DAPI. (D) Representative confocal immunoﬂuorescence images of ASC in THP-1 cells transfected with GFP- ASC or GFP-ASC K174R, infected with http://www.jimmunol.org/ VSV for 3 h. Green represents ASC; blue represents DAPI. (E) ELISA for IL-1b in the supernatant of THP-1 cells transfected with WT ASC or ASC K174R mutant, infected with VSV for 3 h. Cell-based studies were performed at least three times independently with comparable results. Data were presented as mean 6 SEM. Student t test was

used for statistical analysis. Scale bar, by guest on September 24, 2021 10 mm(C and D). **p , 0.01.

also significantly lower than that of WT ASC (Fig. 5E). Taken indicate that MAVS is required for optimal inflammasome acti- together, these results suggested that Lys174, the putative ubiq- vation by RNA virus in vitro. uitination site of ASC via TRAF3, is involved in both aggregates Next, to determine the role of MAVS in the secretion of IL-1b formation and IL-1b induction. during physiologically relevant influenza virus infection in vivo, we challenged mice intranasally with influenza strain A/PR/8, and MAVS is required for optimal inflammasome activation by RNA the amounts of inflammatory cytokines after challenging were virus both in vitro and in vivo measured. IL-1b secretion was evident in serum in WT mice 2 2 Because ASC speck formation leads to caspase-1 activation and infected with 8 3 103 PFU influenza virus. However, MAVS / processing of pro–IL-1b, we thus investigated the role of MAVS in mice failed to secrete a significant amount of IL-1b in response to RNA virus-induced, inflammasome-mediated IL-1b generation, influenza infection (Fig. 6E). Next, we determined the patholog- using A/PR/8 influenza virus, whose ssRNA genome has been ical consequence of inflammasome activation in the infected lung. shown to activate the NLRP3 inflammasome pathways (24, 29). On day 3 postinfection, WT mice showed a spectrum of lesions, IL-1b secretion and caspase-1 activation were observed in WT ranging from single bronchial epithelial cell apoptosis with BMDMs, but were markedly reduced in MAVS2/2 BMDMs, in intraepithelial neutrophil granulocytes to extended bronchial epi- response to RNA virus infection by A/PR/8 influenza (Fig. 6A and thelial cell erosions and lymphocytic peribronchial infiltrates. 2 2 6B). By contrast, MAVS2/2 BMDMs showed normal IL-1b se- In MAVS / mice, the histological lesions at day 3 were cretion and caspase-1 activation in response to crystalline NLRP3 largely limited to single bronchial epithelial cell apoptosis activators MSU and CPPD (Fig. 6B and 6C). Caspase-1 catalysis with few intraepithelial neutrophils and peribronchial lym- activity toward its selective fluorogenic tetrapeptide substrate Ac- phocytes (Fig. 6F). Histology scoring revealed a modest 2 2 YVAD-pNA was also sharply reduced in MAVS2/2 BMDMs in increase of airway inflammation in MAVS / mice, whereas a response to VSV infection (Fig. 6D). Taken together, these results significant increase in airway inflammation was observed in WT 4888 MAVS CONTROLS ASC SPECK FORMATION

FIGURE 6. MAVS is required for optimal inflammasome activation by RNA virus in vitro and in vivo. (A and C) ELISA for IL-1b in the supernatant of WT or MAVS2/2 BMDMs infected with A/PR/8 influenza (MOI = 5) for 6 h (A), treated with MSU (10 mg/ml) for 4 h or CPPD (200 mg/ml) for 4 h (C). (B) Immunoblot showing cleavage of procaspase-1 in WT or MAVS2/2 BMDMs infected with VSV for 3 h, A/PR/8 influenza (MOI = 5) for 6 h, treated with MSU (10 mg/ml) for 4 h or CPPD (200 mg/ml) for 4h.(D) Measurement of caspase-1 activity 2/2 in lysates of WT or MAVS BMDMs Downloaded from infected with VSV for 3 h. (E) ELISA for IL-1b in serum from WT or MAVS2/2 mice infected with A/PR/8 (8 3 103 PFU) for the indicated time. (F) Representative H&E- stained images from lungs of WT or MAVS2/2 mice at 72 h after A/PR/8 (8 3 3 10 PFU) infection. Original magnification http://www.jimmunol.org/ 3200. Cell-based studies were performed at least three times independently with comparable results. Data were presented as mean 6 SEM. Student t test was used for statistical analysis. Numbers below Western blots indicate relative levels determined by software-based quantification of the representative experiment shown. **p , 0.01. by guest on September 24, 2021

mice (Supplemental Fig. 4A). We then examined the role of MAVS of ASC specks. Third, deficiency in MAVS severely impaired in the control of virus infection, and WT and MAVS2/2 mice were IL-1b production. However, because MAVS itself is not an E3 infected intranasally with a sublethal dose (8 3 103 PFU) of A/PR/8 ubiquitin ligase, additional unidentified E3 ligases must be ininfluenza virus. The lungs were collected at the indicated days for volved in this process. determination of viral titers. MAVS2/2 mice tended to have in- Among all E3 ligases that could link MAVS and ASC, TRAF3 creased lung viral titers at day 6 postinfection (Supplemental Fig. was considered as the enzyme regulating ASC turnover. First, it 4B). These results demonstrate that MAVS is responsible for acute specifically interacted with ASC. Second, it formed a complex with lung immunopathology and plays an essential role in limiting virus ASC and contributed to the K63-linked ubiquitination, stabiliza- production. tion, and aggregates formation of ASC upon viral infection. Third, substitution of the conserved cysteine residue of TRAF3 abolished Discussion its E3 ligase activity in ASC ubiquitination. Finally, TRAF3- The CARDs are members of the six-helix bundle death domain- deficient cells exhibited attenuated ASC ubiquitination and fold superfamily that mediate the assembly of large signaling IL-1b production in response to VSV infection. These findings complexes in the apoptotic and inflammatory signaling pathways impact the understanding of the complex relationship between (30). The adaptor ASC enhances conversion of procaspase-1 to its innate immune effectors and the inflammasome signaling events. enzymatically active form through CARD–CARD interactions. Notably, a recent article found that MAVS recruits multiple The RLR pathway adaptor MAVS also contains an N-terminal ubiquitin E3 ligases including TRAF2, 3, 5, and 6 to activate CARD for interaction with RIG-I. In this article, we provide antiviral signaling cascades (31). They found that IFN regulatory evidence that these two adaptors are involved in optimal inflam- factor (IRF) 3 activation was abolished in cells lacking TRAF2, 5, masome function upon RNA virus infection through CARD– and 6. However, TRAF3 is not essential for MAVS signaling. The CARD interaction. Several lines of evidence indicated that function of TRAF3 is still mysterious. We made use of MAVS signaling pathways dependent on MAVS positively regulated mutant (E457D/Q145N), which lost its ability to bind TRAF3, and inflammasome signaling. First, MAVS was required for the sta- found that MAVS engagement of TRAF3 induced by RNA virus bilization of ASC. Second, MAVS was required for the formation infection is critical for ASC speck formation and the subsequent The Journal of Immunology 4889

ASC inflammasome activation. Recent findings also found that exhibited dramatically increased mortality but reduced immune MAVS recruits other proteins including NLRP3 and VDAC1 to response after influenza virus exposure (39). RIG-I is a pivotal participate in inflammasome signaling by noncrystal activators regulator of inflammasome activation ininfluenza A virus–infected and apoptosis induction pathway (22, 32). Overall, these findings primary lung epithelial cells through a MAVS/TRIM25/Riplet- support the central role of MAVS to serve as a platform and dependent type I IFN signaling (27). Thus, MAVS may play an spatiotemporally regulate and integrate multiple innate signaling even more profound role in host inflammation and protection upon pathways in mitochondria. influenza infection. NLRP3 expression is a limiting factor for NLRP3 inflammasome Together, these findings revealed a previously uncharacterized activation. The expression of NLRP3 itself is tightly controlled to function of MAVS in viral-induced inflammasome activation, that restrict its ability to directly recognize microbial-derived sub- is, unstressed cytoplasm contains a mixture of unstable ubiqui- stances (33). In this study, we found the expression of ASC, but tinated and stable K63-ubiquitylated ASC; upon viral-induced not NLRP3, was controlled by MAVS–TRAF3 signaling axis, RIG-I–MAVS activation, a portion of ASC in the cytoplasm because deficiency of MAVS results in almost complete depletion serves as the source for ASC K63-ubiquination modification via of ASC protein levels with unchanged NLRP3 expression. activated TRAF3 E3 ligase, and such ubiquitin modification not Legionella pneumophila has been shown to block the formation of only regulates ASC protein stability, but also provides critical inflammasome complexes by interfering with the transcription of signals for the formation of inflammasome complex. The MAVS– gene-encoding ASC and preventing ASC expression (34, 35). TRAF3–ASC axis therefore defines a new signaling cascade for ASC is considered the key to caspase-1 activation within the “fine-tuning” of the spatiotemporal regulation of innate immunity inflammasome in response to almost all upstream triggers known and inflammatory response. Downloaded from to induce maturation of IL-1b. Our results suggest that MAVS is only required for NLRP3 activation specifically induced by RNA Acknowledgments virus infection. In fact, previous study showed that there was We thank Genhong Cheng (Department of Microbiology, Immunology and a graded dependence on MAVS for caspase-1 activation and IL-1b Molecular Genetics, University of California, Los Angeles, Los Angeles, secretion depending upon the dose and nature of the NLRP3 ac- CA) and Zhijian Chen (Department of Molecular Biology, University of 2/2 tivator used, with poly(I:C), ATP, and nigericin showing maximal Texas Southwestern Medical Center, Dallas, TX) for providing TRAF3 http://www.jimmunol.org/ 2 2 dependence on MAVS [in the order of poly(I:C) . ATP, nigericin] and MAVS / MEFs, respectively. (22). Therefore, we assume that RNA virus activators require MAVS, to form MAVS–TRAF3–ASC complex other than the core Disclosures inflammasome complex. Furthermore, it is surprising to see that The authors have no financial conflicts of interest. MAVS is not required for caspase-1 and IL-1b processing in response to MSU (22), because ASC is nearly invisible in MAVS References knockout cells. We believe that other factors besides MAVS may 1. Bryan, N. B., A. Dorfleutner, Y. Rojanasakul, and C. Stehlik. 2009. Activation of mediate ASC stability. inflammasomes requires intracellular redistribution of the apoptotic speck-like Among the 10 possible MAVS-mediated ubiquitination site(s) in protein containing a caspase recruitment domain. J. Immunol. 182: 3173–3182. by guest on September 24, 2021 ASC, we identified Lys174 of ASC as a critical residue for speck 2. Chen, G., M. H. Shaw, Y. G. Kim, and G. Nun˜ez. 2009. NOD-like receptors: role in innate immunity and inflammatory disease. Annu. Rev. Pathol. 4: 365–398. formation and a possible ubiquitination site. The K174R substi- 3. Villani, A. C., M. Lemire, G. Fortin, E. Louis, M. S. Silverberg, C. Collette, tution in ASC nearly abrogated the ubiquitination of ASC induced N. Baba, C. Libioulle, J. Belaiche, A. Bitton, et al. 2009. Common variants in the NLRP3 region contribute to Crohn’s disease susceptibility. Nat. Genet. 41: 71–76. by VSV infection, which suggested that K174 site served as 4. Zaki, M. H., K. L. Boyd, P. Vogel, M. B. Kastan, M. Lamkanfi, and a physiological regulator of ASC ubiquitination. Notably, viral T. D. Kanneganti. 2010. The NLRP3 inflammasome protects against loss of infection induced the complex formation of WT ASC with TRAF3 epithelial integrity and mortality during experimental colitis. Immunity 32: 379– 391. and MAVS, whereas ASC K174R mutant failed to form a larger 5. Jin, Y., C. M. Mailloux, K. Gowan, S. L. Riccardi, G. LaBerge, D. C. Bennett, complex including both TRAF3 and MAVS. Published studies P. R. Fain, and R. A. Spritz. 2007. NALP1 in vitiligo-associated multiple au- have shown large ASC aggregates localized together with poly- toimmune disease. N. Engl. J. Med. 356: 1216–1225. 6. Martinon, F., V. Pe´trilli, A. Mayor, A. Tardivel, and J. Tschopp. 2006. Gout- ubiquitin under poly(dA:dT) treatment; specifically, ASC itself associated uric acid crystals activate the NALP3 inflammasome. Nature 440: underwent K63-linked ubiquitination (36). The contribution of 237–241. Lys174 site of ASC in inflammasome activation by other activators 7. Magitta, N. F., A. S. Bøe Wolff, S. Johansson, B. Skinningsrud, B. A. Lie, K. M. Myhr, D. E. Undlien, G. Joner, P. R. Njølstad, T. K. Kvien, et al. 2009. A needs further investigation. In addition to the ubiquitination coding polymorphism in NALP1 confers risk for autoimmune Addison’s disease modification of ASC, phosphorylation of ASC has just been re- and type 1 diabetes. Genes Immun. 10: 120–124. 8. Malozowski, S., and J. T. Sahlroot. 2007. Interleukin-1-receptor antagonist in ported to control inflammasome activity through the formation of type 2 diabetes mellitus. N. Engl. J. Med. 357: 302–303, author reply 303. ASC specks (37). The functional relationship between phosphor- 9. Stehlik, C., S. H. Lee, A. Dorfleutner, A. Stassinopoulos, J. Sagara, and ylation and ubiquitination has been widely reported in innate J. C. Reed. 2003. Apoptosis-associated speck-like protein containing a caspase recruitment domain is a regulator of procaspase-1 activation. J. Immunol. 171: immune signaling. For example, Ning et al. (38) demonstrated that 6154–6163. K63-linked ubiquitination of C-terminal residues of IRF7 oc- 10. Duncan, J. A., D. T. Bergstralh, Y. Wang, S. B. Willingham, Z. Ye, curred as a prelude to IRF7 phosphorylation and activation. It is A. G. Zimmermann, and J. P. Ting. 2007. Cryopyrin/NALP3 binds ATP/dATP, is an ATPase, and requires ATP binding to mediate inflammatory signaling. Proc. thus interesting to delineate the exact functional relationship be- Natl. Acad. Sci. USA 104: 8041–8046. tween ubiquitination and phosphorylation of ASC. 11. Faustin, B., L. Lartigue, J. M. Bruey, F. Luciano, E. Sergienko, B. 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TRAF5 is a downstream target of MAVS in antiviral response is also beneficial to the host post influenza in- antiviral innate immune signaling. PLoS ONE 5. Available at: http://journals. fection (40). However, mice lacking NLPR3, ASC, or caspase-1 plos.org/plosone/article?id=10.1371/journal.pone.0009172#pone-0009172-g007. 4890 MAVS CONTROLS ASC SPECK FORMATION

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